Protocol for analyzing root halotropism using split-agar system in Arabidopsis thaliana

Summary Plant roots sense salt gradients in soil to avoid saline environments through halotropism. Here, we present a protocol to study halotropism with an optimized split-agar system that simulates the salt gradient in soil. We describe steps for preparation of the split-agar system, measurement of Na+, and observation of root bending. We then detail segmentation of root cells and visualization of microtubules and cellulose synthases. This system is simple to operate and has broader applications, such as hydrotropism and chemotropism. For complete details on the use and execution of this protocol, please refer to Yu et al. (2022).1


STEP-BY-STEP METHOD DETAILS
Here, we outline the steps for the analysis of Arabidopsis root halotropic response.

Preparing plant materials
Timing: 7-8 days The purpose of these steps is to prepare Arabidopsis seedlings for halotropic treatment.
1. Sterilize the seeds with 5% (v/v) sodium hypochlorite for 10 min in a 1.5 mL Eppendorf tube. 2. Rinse seeds with sterile water for 5 times.
3. Remove the sterilized water. 4. Resuspend the seeds with 0.05% agarose. 5. Prepare 1/2 MS medium (1.2% agar, 1% sucrose, pH 5.7) with a magnetic bar in 1 L bottle and autoclave at 120 C for 20 min. 6. Cool the medium on a magnetic stirrer to about 45 C. 7. Pour autoclaved medium into 10 cm 3 10 cm square disposable Petri dishes (about 40 mL/ plate). 8. Cool down the medium for 15 min and dry for 30-45 min to reduce the water content in the medium and prevent water accumulation during plant growth. 9. Dot the seeds on the plate with a 1 mL pipette.
Note: A plate has 4 rows of seeds, with a spacing of 2 cm between each row. Each row contains about 12-14 seeds spaced about 0.5 cm apart.
10. Seal plates with micropore tape (3M). 11. Wrap the plate with aluminum foil and stratify the seeds at 4 C for 3 days. 12. Grow seedlings vertically in a Percival CU36L5 incubator (or similar) at 21 C-23 C under a 16-h light, 8-h dark photoperiod for 4-5 days.
Preparation of split-agar system with plastic separator Timing: 4-6 h These steps aim to prepare the split-agar plates with a plastic separator.
Note: Sterilize the plastic separator with 75% alcohol for 10 min before use.
16. Seal the junction of the plastic separator and Petri dish with the 1/2 MS medium containing 200 mM NaCl using a 1 mL pipette ( Figure 1B).
Note: The medium flows through the ruler to the other half Petri dish, see the troubleshooting 1.
17. Wait 5 min for the medium to solidify, then pouring the 1/2 MS medium containing 200 mM NaCl into the sealed half Petri dish ( Figures 1C and 1D).

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19. Direct pour the 1/2 MS medium kept in the 65 C-incubator into the empty half of the Petri dish ( Figure 1F).
Note: In this step, other required reagents or compounds can be added to the 1/2 MS medium.
Note: Temperature may affect the solidification time of agar and the diffusion of Na + . Please keep the temperature of 1/2 MS medium as consistent as possible in different batches of experiments.
20. After the medium is solidified (approx. 15 min), and dry it for 30-45 min without the cover.
Note: If water accumulated at the bottom of the square Petri dish, see the troubleshooting 3.
CRITICAL: Considering the diffusion of Na + . The split-agar plates need to be used within 6 h.
Preparation of split-agar system by removing the medium

Timing: 4-6 h
These steps aim to prepare the split-agar plates by cutting the medium.
21. Prepare 1/2 MS medium (1.2% agar, 1% sucrose, pH 5.7) and 1/2 MS medium containing 200 mM NaCl (1.2% agar, 1% sucrose, pH 5.7) with a magnetic bar in 1 L bottle and autoclave at 120 C for 20 min. 22. Place the 1/2 MS medium containing 200 mM NaCl on a magnetic stirrer to cool to 45 C. Keep the 1/2 MS medium in a 65 C incubator. 23. Pour the 1/2 MS medium containing 200 mM NaCl into a 10 cm 3 10 cm square disposable Petri dish (Figures 2A and 2B). 24. Wait for 20 min until the medium solidified, and cut the medium diagonally with a blade (Figure 2C). 25. Press the half-medium inward along the interface between the medium and the Petri dish with the blade, and then press the half-medium out along the cut line ( Figures 2D and 2E). 26. Pour the 1/2 MS medium that was kept in the 65 C incubator into the empty half of the Petri dish ( Figure 2F).
Note: In this step, other required reagents or compounds can be added to the 1/2 MS medium.
Note: Since temperature may affect the solidification time of agar and the diffusion of Na + . Please keep the temperature of 1/2 MS medium as consistent as possible in different batches of experiments.
27. After the medium is solidified for 15 min, dry it for 30-45 min without cover.
Note: If water accumulated at the bottom of the square Petri dish, see the troubleshooting 3.
CRITICAL: Considering the diffusion of Na + . Split-agar plates need to be used within 6 h. These steps aim to analyze the root halotropism of Arabidopsis seedlings. c. Transfer 10-12 seedlings to a Petri dish, with an interval of 0.5-0.75 cm for each seedling ( Figure 3B).

Note:
If there are different kinds of genotypes, it is better to put them on the same split-agar plate ( Figures 3C and 3D).
Note: Reagents or compounds can be added to 1/2 MS medium to test their effects on halotropism ( Figures 4A and 4D).
30. Seal the Petri dishes with micropore tape, and put back the treated seedlings into the same culture environment for vertical growth. 31. Visualize the microtubule dynamics after growing for about 1-2 h. Photograph the root growth after 10-16 h of growth.
Note: If the root stops growing after salt avoidance treatment, see the troubleshooting 4.

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Visualization of Na + in Arabidopsis root tips

Timing: 4-8 h
These steps aim to visualize Na + with CoroNa Green.
32. Prepare 4-5 day-old vertically growing seedlings according to steps 1-12. 33. Transfer the seedlings to a split-agar plate with halostimulation for 3 h according to steps 28-31. 34. Rinse the treated seedlings three times in sterilized water quickly (about 1 s each).
Note: This step is mainly to wash the excess Na + adsorbed on the root surface.
35. Stain the seedlings with 20 mM CoroNa Green (CoroNa Green Sodium Indicator, Thermo Fisher, C36676) for 1 h. 36. Rinse the stained seedlings three times in sterilized water quickly (about 1 s each).
Note: This step is mainly to wash the excess CoroNa Green adsorbed on the root surface. Note: If the fluorescence is too strong after dye application, see the troubleshooting 5.

Prepare plant materials for observation of root bending under a microscope
Timing: 6-7 days These steps aim to prepare the plant materials.

Observation of root bending under a microscope
Timing: 5-6 h These steps aim to observe root epidermal cell expansion during root bending.
45. Pour 1/2 MS medium into a chambered cover slide (ibidi, 80287), and make sure to fill the entire chamber by covering it with a sterile glass plate.  Figure 5A). 54. Select a detector: MorphoLibJ detector, and select next. 55. Check ''Simplify contours'', select preview, and get a preliminary result with the setting. Adjust to outline most of the cells and select next ( Figure 5B).
Note: Use the recommended Tolerance and Connectivity for the first time.
Note: Increase the Tolerance to get less detail.
56. Leave Initial thresholding as it is and select next. 57. Set filters on spots as area and check Below to cover only the cells ( Figure 5C). 58. Select Overlap tracker.
59. Edit settings at Display options to get preferred display (set spot alpha transparency to 1) (Figure 5D). 60. At ''Select an action'', select Capture overlay, and select execute to export the segmented images.
Note: The finalized images are in one stack, with each cell colored differently. In some cases, the algorithm may not recognize cells as the same cell at different time points, then change the color manually using Flood Fill Tool.
Prepare plant seedlings for observation of cortical microtubules and CESAs in root epidermal cells

Timing: 3-4 days
These steps aim to prepare the plant seedlings.
61. Prepare chambered cover slides (ibidi, 80287) with about 2 mm-thick 1/2 MS, 1% sucrose, and 1.2% agar (adjust pH to 5.7 before autoclave) in each chamber ( Figure 6A). 62. Sterilize homozygous transgenic seeds with 5% sodium hypochlorite for 10 min in a 1.5 mL Eppendorf tube. 63. Rinse seeds in sterile MilliQ water 5 times. 64. Sow seeds on the right side of 1/2 MS in chambered cover slides with 0.3 cm intervals, and push the seeds into the medium until they are close to the cover slides in the bottom. 65. Seal chambered cover slides with micropore tape. 66. Stratify the seeds at 4 C for 2 days in the dark. Note: Detect GFP signals with a 488-nm laser and a 482/35-nm emission filter, and mCherry signals with a 561-nm laser and a 542/27-nm emission filter, respectively.

Observation of cortical microtubules and CESAs in root epidermal cells
CRITICAL: Since gravity affects the dynamics of microtubules, a vertical platform is required to avoid gravity interference.

EXPECTED OUTCOMES
In the split-agar system, halotropic growth of root tips can be observed in 2-6 h. Therefore, this split-agar system is suitable for screening early salt signaling elements in genetic screens. In addition, we established a split-agar system suitable for microscopy with the chambered cover slides. This is helpful for real-time monitoring of cytological processes during the early halotropic response, such as calcium signals, ROS accumulation, cytoskeleton dynamics, hormone distribution, etc (Figure 7).

LIMITATIONS
Since exogenous reagents need to be added to 1/2 MS at about 65 C, our method is not suitable for reagents or compounds that are thermally unstable.

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Importantly, our method cannot completely simulate the soil environment, and other experimental methods are needed to realize the response of the change of root morphology in the soil.
Note: The interface between mediums may form mechanical stimulation. The medium flows through the ruler to the other half Petri dish (related to step 16).

Potential solution
Before pouring the medium, check whether the connection between the ruler and the Petri dish is completely sealed.

Problem 2
When removing the ruler, it is prone to stick the medium on the ruler, resulting in an uneven section of the medium (related to step 18).

Potential solution
It may be that the AGAR is not completely solidified, and the solidification time can be increased by 5-10 min in step 18. And do not pull up the ruler when removing it, but quickly push the ruler toward the empty half of the Petri dish.

Problem 3
Water accumulated at the bottom of the square Petri dish (related to steps 20 and 27). This will make the surface of medium water soaked and affect the attachment of the root, thus affecting root halotropic growth.

Potential solution
Increase the drying time when preparing the split-agar plates. We suggest that the additional drying time should not exceed 30 min, and the drying time should be consistent for each experiment.

Problem 4
The root stops growing after salt avoidance treatment (related to step 31).

Potential solution
The ruler and blade may contain some compounds that have not been removed and affect the root growth. Before using the ruler and blade, wash them with sterile water three times.

Problem 5
The fluorescence is too strong after dye application (related to step 37).

Potential solution
Increase the washing times in step 36 and reduce the CoroNa Green concentration (It can be as low as 1 mM).

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Yang Zhao (yangzhao@psc.ac.cn).

Materials availability
All the non-commercial materials described in this study are available upon request.

Data and code availability
This study did not generate/analyze datasets.